Genetic analysis in HUDEP-2 cells reveals that GYPA and GYPC are the primary determinants of EBA-175 and EBA-140 binding to cRBCs, respectively. (A) Schematic of HUDEP-2 proliferation and differentiation protocol, and representative images of differentiating HUDEP-2 cells at ×100 magnification. Day 2, proerythroblasts; day 4, polychromatic erythroblasts; and day 6, orthos. (B) Flow cytometry–based binding assays of RII regions of EBA-175-His (left) and EBA-140-His (right) incubated with WT vs CD44-null HUDEP-2 orthos. Binding of the EBA proteins to the cells was quantified using anti-His antibody and fluorescent secondary antibody. (C) Flow cytometry–based binding assays of RII regions of EBA-175-His (left) and EBA-140-His (right) incubated with WT, GYPA-null, or GYPC-null HUDEP-2 orthos. Binding was quantified using a mouse anti–6x-His antibody (Invitrogen; 1:300) followed by goat anti-mouse IgG-FITC (1:1000). (D) Flow cytometry–based binding assays of RII regions of EBA-175-His (left) and EBA-140-His (right) incubated with WT, GYPA-null, GYPA/CD44-null, GYPC-null, or GYPC/CD44-null HUDEP-2 orthos. Binding was quantified using the anti–6x-His antibody and fluorescent secondary antibody. DEX, dexamethasone; DOX, doxycycline.